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Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Treatment of RMS usually includes primary tumor resection along with systemic chemotherapy. Two-dimensional (2D) cell culture systems and animal models have been extensively used for investigating the potential efficacy of new RMS treatments. However, RMS cells behave differently in 2D culture than in vivo, which has recently inspired the adoption of three-dimensional (3D) culture environments. In the current paper, we will describe the detailed methodology we have developed for fabricating a 3D engineered model to study alveolar RMS (ARMS) in vitro. This model consists of a thermally cross-linked collagen disk laden with RMS cells that mimics the structural and bio-chemical aspects of the tumor extracellular matrix (ECM). This process is highly reproducible and produces a 3D engineered model that can be used to analyze the cytotoxicity and autophagy induction of drugs on ARMS cells. The most improtant bullet points are as following:•We fabricated 3D model of ARMS.•The current ARMS 3D model can be used for screening of chemotherapy drugs.•We developed methods to detect apoptosis and autophagy in ARMS 3D model to detect the mechansims of chemotherapy agents., Graphical abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue malignant tumor. Despite numerous clinical trials that have investigated the efficacy of multidrug chemotherapy regimens, outcomes for high-risk RMS patients are still poor which has inspired research into new targeted therapies that molecularly target malignant RMS cells. In order to evaluate such therapies in vitro in a realistic tumor microenvironment, we have bioengineered a cell-laden 3D tissue-engineered model with uniform geometry and an uncomplicated and reproducible biofabrication process. The transparent collagen-I hydrogel promotes cell attachment and growth and allows for analysis of the cellular response to drugs through brightfield microscopy, immunocytochemistry, and live/dead viability assays.Image, graphical abstract

Glioblastoma multiforme (GBM) is a rapidly progressive and deadly form of brain tumor with a median survival rate of ~15 months. GBMs are hard to treat and significantly affect the patient's physical and cognitive abilities and quality of life. Temozolomide (TMZ)&mdash, an alkylating agent that causes DNA damage&mdash, is the only chemotherapy choice for the treatment of GBM. However, TMZ also induces autophagy and causes tumor cell resistance and thus fails to improve the survival rate among patients. Here, we studied the drug-induced programmed cell death and invasion inhibition capacity of TMZ and a mevalonate cascade inhibitor, simvastatin (Simva), in a three-dimensional (3D) microfluidic model of GBM. We elucidate the role of autophagy in apoptotic cell death by comparing apoptosis in autophagy knockdown cells (Atg7 KD) against their scrambled counterparts. Our results show that the cells were significantly less sensitive to drugs in the 3D model as compared to monolayer culture systems. An immunofluorescence analysis confirmed that apoptosis is the mechanism of cell death in TMZ- and Simva-treated glioma cells. However, the induction of apoptosis in the 3D model is significantly lower than in monolayer cultures. We have also shown that autophagy inhibition (Atg7 KD) did not change TMZ and Simva-induced apoptosis in the 3D microfluidic model. Overall, for the first time in this study we have established the simultaneous detection of drug induced apoptosis and autophagy in a 3D microfluidic model of GBM. Our study presents a potential ex vivo platform for developing novel therapeutic strategies tailored toward disrupting key molecular pathways involved in programmed cell death and tumor invasion in glioblastoma.

A continuous nonlinear full-state time-delayed feedback control scheme is designed within the framework of geometric mechanics to stabilize the rigid body attitude motion, which is subject to an unknown constant time delay in feedback measurement. The attitude kinematics is globally described on the matrix Lie group S O ( 3 ) of rigid body rotations. A Morse–Lyapunov–Krasovskii functional is utilized to guarantee the asymptotic stability of the system, which yields the control gain matrices via linear matrix inequality stability conditions. In light of this result, a delayed feedback control scheme is also designed for the planar rotational motion to examine the almost global asymptotic stability of the system in the presence of an unknown time delay in feedback measurement. Simulations are performed for the proposed control schemes based on the discretized models of the controlled systems and the performance of the proposed controllers are evaluated by employing large maneuvers.

An integrated guidance and feedback control scheme for steering an underactuated vehicle through desired waypoints in three-dimensional space, is developed here. The underactuated vehicle is modeled as a rigid body with four control inputs. These control inputs actuate the three degrees of freedom of rotational motion and one degree of freedom of translational motion in a vehicle body-fixed coordinate frame. This actuation model is appropriate for a wide range of underactuated vehicles including spacecraft with internal attitude actuators, vertical take-off and landing (VTOL) aircraft, fixed-wing multirotor unmanned aerial vehicles (UAVs), maneuverable robotic vehicles, etc. The guidance problem is developed on the special Euclidean group of rigid body motions, SE(3), in the framework ofgeometric mechanics, which represents the vehicle dynamics globally on this configuration manifold. The integrated guidance and control algorithm selects the desired trajectory for the translational motion that passes through the given waypoints, and the desired trajectory for the attitude based on the desired thrust direction to achieve the translational motion trajectory. A feedback control law is then obtained to steer the underactuated vehicle towards the desired trajectories in translation and rotation. This integrated guidance and control scheme takes into account known bounds on control inputs and generates a trajectory that is continuous and at least twice differentiable, which can be implemented with continuous and bounded control inputs. The integrated guidance and feedback control scheme is applied to an underactuated quadcopter UAV to autonomously generate a trajectory through a series of given waypoints in SE(3) and track the desired trajectory in finite time. The overall stability analysis of the feedback system is addressed. Discrete time models for the dynamics and control schemes of the UAV are obtained in the form of Lie group variational integrators using the discrete Lagrange-d’Alembert principle. Almost global asymptotic stability of the feedback system over its state space is shown analytically and verified through numerical simulations.

Statins are some of the most widely used drugs worldwide, but one of their major side effects is myotoxicity. Using mouse myoblast (C2C12) and human alveolar rhabdomyosarcoma cell lines (RH30) in both 2-dimensional (2D) and 3-dimensional (3D) cell culture, we investigated the mechanisms of simvastatin's myotoxicity. We found that simvastatin significantly reduced cell viability in C2C12 cells compared to RH30 cells. However, simvastatin induced greater apoptosis in RH30 compared to C2C12 cells. Simvastatin-induced cell death is dependent on geranylgeranyl pyrophosphate (GGPP) in C2C12 cells, while in RH30 cells it is dependent on both farnesyl pyrophosphate (FPP) and GGPP. Simvastatin inhibited autophagy flux in both C2C12 and RH30 cells and inhibited lysosomal acidification in C2C12 cells, while autophagy inhibition with Bafilomycin-A1 increased simvastatin myotoxicity in both cell lines. Simvastatin induced greater cell death in RH30 cells compared to C2C12 in a 3D culture model with similar effects on autophagy flux as in 2D culture. Overall, our results suggest that simvastatin-induced myotoxicity involves both apoptosis and autophagy, where autophagy serves a pro-survival role in both cell lines. The sensitivity to simvastatin-induced myotoxicity differs between 2D and 3D culture, demonstrating that the cellular microenvironment is a critical factor in regulating simvastatin-induced cell death in myoblasts.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

Rhabdomyosarcoma (RMS) is a muscle-derived tumor. In both pre-clinical and clinical studies Temozolomide (TMZ) has been recently tested against RMS; however, the precise mechanism of action of TMZ in RMS remains unclear. Here we demonstrate that TMZ decreases the cell viability of the RH30 RMS and C2C12 cell line, where cells display evidence of mitochondrial outer membrane permeability. Interestingly, the C2C12 mouse myoblast line was relatively more resistant to TMZ-induced apoptosis. Moreover, we observed that TMZ activated biochemical and morphological markers of autophagy in both cell lines. Autophagy inhibition in both RH30 and C2C12 cells significantly increased TMZ-induced cell death. In RH30 cells, TMZ increased Mcl-1 and Bax protein expression compared to corresponding time match controls while in C2C12 Mcl-1, Bcl-2, Bcl-XL, and Bax protein expression were not changed. Baf-A1 co-treatment with TMZ significantly decrease Mcl-1 expression compared to TMZ while increase Bax expression in C2C12 cells (Bcl2 and Bcl-XL do not significantly change in Baf-A1/TMZ co-treatment). Using a three-dimensional (3D) C2C12 and RH30 culture model we demonstrated that TMZ is significantly more toxic in RH30 cells (live/dead assay). Additionally, we have observed in our 3D culture model that TMZ induced both apoptosis (cleavage of PARP) and autophagy (LC3-puncta and localization of LC3/p62). Therefore, our data demonstrate that TMZ induces simultaneous autophagy and apoptosis in both RH30 and C2C12 cells in 2D and 3D culture model, where RH30 cells are more sensitive to TMZ-induced death. Furthermore, autophagy serves to protect RH30 cells from TMZ-induced death.

In this article, we present a method for cell culture on a digital microfluidic (DMF) platform. Dielectrophoresis is used for trapping and patterning cells encapsulated within a 3D gelatin methacrylate (GelMA) hydrogel. In this method, planar traps are patterned within the electrodes on the DMF chip. Dispersed HEK 293 cells in GelMA solution are used to show negative dielectrophoresis when subject to a voltage at 20 kHz. The effects of the cell concentration, trap area, and trap spacing are analyzed. The rise time for patterning cells is shown to be rapid, from 8.5 seconds to 198.3 seconds for 50 μm and 500 μm diameter traps, respectively. The number of cells trapped was significantly influenced by the trap diameter and cell concentration. Live/dead assayed cells under a fluorescence microscope indicated that clusters of HEK 293 cells are 66% viable after 4 days of culturing. Certain trap designs were 78% viable on average after 4 days. This platform can be applied to execute quantitative, automated cell behavior studies and drug tests on the array of cells in vitro.

Following the development of microfluidic systems, there has been a high tendency towards developing lab-on-a-chip devices for biochemical applications. A great deal of effort has been devoted to improve and advance these devices with the goal of performing complete sets of biochemical assays on the device and possibly developing portable platforms for point of care applications. Among the different microfluidic systems used for such a purpose, digital microfluidics (DMF) shows high flexibility and capability of performing multiplex and parallel biochemical operations, and hence, has been considered as a suitable candidate for lab-on-a-chip applications. In this review, we discuss the most recent advances in the DMF platforms, and evaluate the feasibility of developing multifunctional packages for performing complete sets of processes of biochemical assays, particularly for point-of-care applications. The progress in the development of DMF systems is reviewed from eight different aspects, including device fabrication, basic fluidic operations, automation, manipulation of biological samples, advanced operations, detection, biological applications, and finally, packaging and portability of the DMF devices. Success in developing the lab-on-a-chip DMF devices will be concluded based on the advances achieved in each of these aspects.

The delayed feedback stabilization of rigid spacecraft attitude dynamics in the presence of an unknown time-varying delay in the measurement is addressed. The attitude representation is parameterized using minimal attitude coordinates. The time-varying delay and its derivative are assumed to be bounded. By employing a linear state feedback controller via a Lyapunov–Krasovskii functional, a general delay-dependent stability condition is characterized for the closed-loop parameterized system in terms of a linear matrix inequality (LMI) whose solution gives the suitable controller gains. An estimate of the region of attraction of the controlled system is also obtained, inside which the asymptotic stability of parameterized system is guaranteed.

The delayed feedback control of rigid body attitude motion is addressed where there is an unknown time delay in the feedback measurement. The attitude motion is described on the tangent bundle TSO(3) to globally and uniquely represent the orientation of the rigid body, while a continuous nonlinear delayed feedback control law is proposed for local asymptotic stabilization on TSO(3). For this purpose, we introduce a notion based on the Lyapunov technique, which combines the Morse-Lyapunov method with Lyapunov-Krasovskii technique to yield a suitable Morse-Lyapunov-Krasovskii (M-L–K) functional, from which the stability conditions and proper control gain matrices are obtained in terms of linear matrix inequalities (LMIs). Simulations illustrate the performance of the proposed control scheme.

In the present study, a hydrodynamic particle manipulation method for digital microfluidic platforms is introduced to separate and concentrate non-buoyant particles in micro-droplets. The proposed method utilizes the combined effects of the gravitational forces and the fluid flow inside the droplet, and it depends only on the hydrodynamic properties of the system without the need of external electrical or magnetic modules. The desired hydrodynamic effect is created by manipulating the droplet in a controlled fashion on a circular pattern of electrodes using the electrowetting-on-dielectric technique. Two successful electrode designs (Star Design and Square Design) are presented. To investigate the feasibility of the proposed particle separation technique, suspensions of silica and polystyrene beads are tested. The fabricated devices successfully focus the non-buoyant silica beads of 1 μm and 5 μm and polystyrene beads of 15 μm in a region on the central electrode; whereas the focusing behavior is not observed for the polystyrene beads of 5 μm. A capturing efficiency of 86% and 94% is achieved for the 5 μm silica particles and 15 μm polystyrene particles, respectively. A parametric study is conducted to investigate the dependency of the focusing regimes on the particle size and density, the droplet size and the electrode geometry, and the actuation scheme. To understand the focusing mechanism, the fluid flow inside the droplet is simulated numerically, and the observed time scales are verified through analytical calculations. Finally, the application of the current particle focusing technique is illustrated for detection of the presence of a low concentration of DNA (18 μg mL−1).

In this paper, a numerical approach is developed for the stability analysis of linear stochastic delay differential equations (SDDEs) in the parameter space based on the Chebyshev Spectral Continuous Time Approximation (CSCTA) technique. The CSCTA method is used to approximate an infinite-dimensional linear SDDE as a set of linear stochastic differential equations (SDEs). The mean and mean-square stability concepts are employed for the stochastic stability analysis of the resulting SDE. For this purpose, a set of linear deterministic differential equations for both the first and second moments are obtained using the Ito differential rule. Two examples are provided: a first order SDDE with multiplicative stochastic excitation and a second order SDDE with both additive and multiplicative stochastic excitation. In both examples the stability charts obtained from the proposed approach match those obtained using the stochastic semi-discretization method as described by Elbeyli et al. (Commun Nonlinear Sci Numer Simul 10(1):85–94, 2005). In the first example the stability results obtained from both numerical approaches are found to be less conservative than the Lyapunov-based stability region obtained by Samiei et al. (Int J Dyn Control 1(1):64–80, 2013).

In this paper, we obtain an analytical Lyapunov- based stability conditions for scalar linear and nonlinear stochastic systems with discrete time-delay. The Lyapunov–Krasovskii and Lyapunov–Razumikhin methods are applied with techniques from stochastic calculus to obtain the regions of mean square asymptotic stability in the parameter space. Both delay-independent and delay-dependent stability conditions are analyzed corresponding to both additive and multiplicative stochastic Brownian motion excitation in the Ito form. It is also shown that the derived sufficient conditions are less conservative in comparison with other numerical LMI-based Lyapunov approaches. A range of different stability charts are obtained based on the derived Lyapunov-based stability criteria, which are also compared with numerical first and second moment stability boundaries computed using the stochastic semidiscretization method. A Lipschitz condition is used to treat nonlinear functions of the current and delayed states.

A new algorithm named random particle optimization algorithm (RPOA) for local path planning problem of mobile robots in dynamic and unknown environments is proposed. The new algorithm inspired from bacterial foraging technique is based on particles which are randomly distributed around a robot. These particles search the optimal path toward the target position while avoiding the moving obstacles by getting help from the robot’s sensors. The criterion of optimal path selection relies on the particles distance to target and Gaussian cost function assign to detected obstacles. Then, a high level decision making strategy will decide to select best mobile robot path among the proceeded particles, and finally a low level decision control provides a control signal for control of considered holonomic mobile robot. This process is implemented without requirement to tuning algorithm or complex calculation, and furthermore, it is independent from gradient base methods such as heuristic (artificial potential field) methods. Therefore, in this paper, the problem of local mobile path planning is free from getting stuck in local minima and is easy computed. To evaluate the proposed algorithm, some simulations in three various scenarios are performed and results are compared by the artificial potential field.

Several studies have been performed on the integration of biosensors into digital microfluidics (DMF). Despite the general success in their detection capabilities, there are still two challenges associated with the integration of biosensors into DMF: (1) complete removal of the droplet containing the analytes from the sensing surface; and (2) biochemical regeneration of the biosensor involving detaching the target analyte from the receptor after each round of sensing. The latter is case dependent and the solution can vary from one application to another. Our research aims at addressing the former, the solution to which is applicable to all biosensors integrated to DMF. This paper presents a thorough characterization of the hydrophilic surface of the biosensor in terms of wettability and geometry, taking into account the overall configuration of the DMF platform. Consequently, we identify the optimal geometry of the sensing surface and the DMF platform providing successful removal of the target droplet from the sensing surface after detection. Based on the results, the gap height is suggested to be chosen at the upper limit of the applicable range. Also, the biosensor, patterned on the device top plate, is recommended to be designed with a high aspect ratio and aligned with the center of the actuating electrode. As a proof of concept, the optimum configuration is implemented on a DMF platform with an interdigitated capacitive biosensor to detect different concentrations of Cryptosporidium, for which it is shown that the sample droplet is removed successfully from the superhydrophilic surface of the biosensor.